Search

The viral responsive protein 15 from the black tiger shrimp Penaeus monodon (PmVRP15) is a highly responsive gene upon white spot syndrome virus (WSSV) challenge. It is identified from hemocyte and important for WSSV trafficking and assembly. However, the knowledge of PmVRP15 gene regulation is limited. In the present study, the genome organization and 5'upstream promoter sequences of PmVRP15 gene were investigated. The PmVRP15 gene was found to contain 4 exons interrupted by 3 introns and the start codon was located in the exon 2. The transcription start site and TATA box were also determined from the 5' upstream sequence. By using the narrow down experiment, the 5' upstream promoter active region was determined to be at the nucleotide positions -525 to +612. Mutagenesis of the putative transcription factor (TF) binding sites revealed that the binding site of interferon regulatory factor (IRF) (-495/-479) was a repressor-binding site whereas those of the octamer transcription factor 1 (Oct-1) (-275/-268) and the nuclear factor of activated T-cells transcription factor (NFAT) (-228/-223) were activator-binding sites. This is the first report on the transcription factors that might play essential roles in modulating the PmVRP15 gene expression. Nevertheless, the underlying regulation mechanism of PmVRP15 gene expression needs further investigation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Plasmolipin has been characterized as a cell entry receptor for mouse endogenous retrovirus. In black tiger shrimp, two isoforms of plasmolipin genes, PmPLP1 and PmPLP2, have been identified from the Penaeus monodon EST database. The PmPLP1 is highly up-regulated in yellow head virus (YHV)-infected shrimp. Herein, the function of PmPLP1 is shown to be involved in YHV infection. The immunoblotting and immunolocalization showed that the PmPLP1 protein was highly expressed and located at the plasma membrane of gills from YHV-infected shrimp. Moreover, the PmPLP1 expressed in the Sf9 insect cells resided at the cell membrane rendering the cells more susceptible to YHV infection. Using the ELISA binding and mortality assays, the synthetic external loop of PmPLP1 was shown to bind the purified YHV and neutralize the virus resulting in the decrease in YHV infection. Our results suggested that the PmPLP1 was likely a receptor of YHV in shrimp., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific strain of Vibrio parahaemolyticus (VP AHPND ), results in great loss of global shrimp production. Despite this, studies on shrimp defense mechanisms protecting against AHPND are few. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes from white shrimp Litopenaeus vannamei hepatopancreas upon VP AHPND infection at the early stages: 3 and 6 h post challenge and in the late stage at 48 h post challenge. Hemocyanin (HMC) is the most abundant gene identified as the up-regulated gene in the SSH library. Various hemocyanin subunits such as hemocyanin (HMC), hemocyanin subunit L1 (HMCL1), L2 (HMCL2), L3 (HMCL3), and L4 (HMCL4) were analyzed for their expression levels upon VP AHPND infection and in response to challenge with partially purified toxin of VP AHPND by qRT-PCR. Only HMC was highly up-regulated at 3 and 6 h post challenge in response to VP AHPND challenge. Two HMC subunits, HMCL3 and HMCL4, were up-regulated in the early phase of VP AHPND toxin injection. Furthermore, all subunits were down-regulated in the late phase of VP AHPND and toxin challenges. The native hemocyanin protein purified from shrimp hemolymph, identified as mixture of HMC and HMCL1, exhibited agglutination activity on VP AHPND . Injecting the purified native hemocyanin along with VP AHPND into shrimp decreased the number of bacteria in the hemolymph as compared to the VP AHPND challenged control. Moreover, pre-incubation of the purified native hemocyanin and VP AHPND toxin prior to injection into shrimp resulted in the decrease of cumulative mortality of shrimp when compared to the control. In addition, protein-protein interaction analysis carried out by ELISA technique indicated that hemocyanin exhibited VP AHPND toxin-neutralizing activity through direct interaction with PirA subunit with a dissociation constant of 6.83 × 10 -6  M. Our results indicated that upon VP AHPND infection the expression of hemocyanin was induced and hemocyanin functions might involve agglutination of invading VP AHPND and also neutralization of VP AHPND secreted toxin via direct interacting with the PirA protein., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Diseases have caused tremendous economic losses and become the major problem threatening the sustainable development of shrimp aquaculture. The knowledge of host defense mechanisms against invading pathogens is essential for the implementation of efficient strategies to prevent disease outbreaks. Like other invertebrates, shrimp rely on the innate immune system to defend themselves against a range of microbes by recognizing and destroying them through cellular and humoral immune responses. Detection of microbial pathogens triggers the signal transduction pathways including the NF-κB signaling, Toll and Imd pathways, resulting in the activation of genes involved in host defense responses. In this review, we update the discovery of components of the Toll and Imd pathways in shrimp and their participation in the regulation of shrimp antimicrobial peptide (AMP) synthesis. We also focus on a recent progress on the two most powerful and the best-studied shrimp humoral responses: AMPs and melanization. Shrimp AMPs are mainly cationic peptides with sequence diversity which endues them the broad range of activities against microorganisms. Melanization, regulated by the prophenoloxidase activating cascade, also plays a crucial role in killing and sequestration of invading pathogens. The progress and emerging research on mechanisms and functional characterization of components of these two indispensable humoral responses in shrimp immunity are summarized and discussed. Interestingly, the pattern recognition protein (PRP) crosstalk is evidenced between the proPO activating cascade and the AMP synthesis pathways in shrimp, which enables the innate immune system to build up efficient immune responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The viral responsive protein 15 from black tiger shrimp Penaeus monodon (PmVRP15), is highly up-regulated and produced in the hemocytes of shrimp with white spot syndrome virus (WSSV) infection. To investigate the differential expression of genes from P. monodon hemocytes that are involved in WSSV infection under the influence of PmVRP15 expression, suppression subtractive hybridization (SSH) of PmVRP15-silenced shrimp infected with WSSV was performed. The 189 cDNA clones of the forward library were generated by subtracting the cDNAs from WSSV-infected and PmVRP15 knockdown shrimp with cDNAs from WSSV-infected and GFP knockdown shrimp. For the opposite subtraction, the 176 cDNA clones in the reverse library was an alternative set of genes in WSSV-infected shrimp hemocytes in the presence of PmVRP15 expression. The abundant genes in forward SSH library had a defense/homeostasis of 26%, energy/metabolism of 23% and in the reverse SSH library a hypothetical protein with unknown function was found (30%). The differential expressed immune-related genes from each library were selected for expression analysis using qRT-PCR. All selected genes from the forward library showed high up-regulation in the WSSV-challenged PmVRP15 knockdown group as expected. Interestingly, PmHHAP, a hemocyte homeostasis associated protein, and granulin-like protein, a conserved growth factor, are extremely up-regulated in the absence of PmVRP15 expression in WSSV-infected shrimp. Only transcript level of transglutaminase II, that functions in regulating hematopoietic tissue differentiation and inhibits mature hemocyte production in shrimp, was obviously down-regulated as observed from SSH results. Taken together, our results suggest that PmVRP15 might have a function relevant to hemocyte homeostasis during WSSV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

ALFPm6, a member of antimicrobial peptide in the antilipopolysaccharide factor (ALF) family from Penaeus monodon, plays important roles in shrimp immunity against pathogens. However, its antimicrobial activity and underlying mechanism have not been reported. The synthetic cyclic ALFPm6#29-52 peptide (cALFPm6#29-52) corresponding to the ALFPm6 LPS-binding domain can agglutinate and exhibited bacterial killing activity toward a Gram-negative bacterium, Escherichia coli 363 and Gram-positive bacteria, Bacillus megaterium, Aerococcus viridans, and Micrococcus luteus, with MIC values of 25-50 μM. Specifically, ALFPm6 and ALFPm3, the most abundant ALF isoforms, are different in terms of gene expression patterns upon pathogen infections. Herein, the regulation of ALFPm3 and ALFPm6 gene expression was studied. The 5'-upstream and promoter sequences were identified and the putative transcription factor (TF)-binding sites were predicted. The narrow down assay indicated that the ALFPm3 promoter and partial promoter of the ALFPm6 active regions were located at nucleotide positions (-814/+302) and (-282/+85), respectively. Mutagenesis of selected TF-binding sites revealed that Rel/NF-κB (-280/-270) of ALFPm3 and C/EBPβ (-88/-78) and Sp1 (-249/-238) sites of ALFPm6 were the activator-binding sites. Knockdown of the PmMyD88 and PmRelish genes in V. harveyi-infected shrimp suggested that the ALFPm3 gene was regulated by Toll and IMD pathways, while the ALFPm6 gene was regulated by the Toll pathway.

In innate immunity, antimicrobial peptides (AMPs) play a vital role in combating microbial pathogens. Among the AMPs identified in Penaeus monodon, only anti-lipopolysaccharide factor isoform 3 (ALFPm3) has been reported to exhibit activity against white spot syndrome virus (WSSV). However, the mechanism(s) involved are still not clear. In the present study, ALFPm3-interacting proteins were screened for from a WSSV library using the yeast two-hybrid screening system, revealing the five potential ALFPm3-interacting proteins of WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471. Temporal transcriptional analysis in WSSV-infected P. monodon revealed that all five of these WSSV gene transcripts were expressed in the late phase of infection (24h and 48h post-infection). Of these, WSSV189 that was previously identified as a structural protein, was selected for further analysis and was shown to be an enveloped protein by Western blot and immunoelectron microscopy analyses. The in vitro pull-down assay using recombinant WSSV189 (rWSSV189) protein as bait confirmed the interaction between ALFPm3 and WSSV189 proteins. Moreover, pre-incubation of rWSSV189 protein with rALFPm3 protein interfered with the latter's neutralization effect on WSSV in vivo, as shown by the increased cumulative mortality of shrimp injected with WSSV following prior treatment with pre-incubated rWSSV189 and rALFPm3 proteins compared to that in shrimp pre-treated with rALFPm3 protein. Thus, ALFPm3 likely performs its anti-WSSV action by binding to the envelope protein WSSV189 and possibly other WSSV structural proteins., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Serpin or serine proteinase inhibitor is a family of protease inhibitors that are involved in controlling the proteolytic cascade in various biological processes. In shrimp, several serpins have been identified but only a few have been characterized. Herein, the PmSERPIN3 gene identified from Penaeus monodon EST database was studied. By using the 5'- and 3'-Rapid Amplification of cDNA Ends (RACE) techniques, the full-length of PmSERPIN3 cDNA was obtained. The cDNA contained an open reading frame of 1233 bp encoding for 410 amino acid residue protein. Genome sequence analysis revealed that the PmSERPIN3 was an intronless gene. RT-PCR analysis revealed that it was constitutively expressed in all developmental stages, all shrimp tissues tested, and upon pathogen infections. The recombinant mature PmSERPIN3 protein (rPmSERPIN3) produced in Escherichia coli exhibited inhibitory activity against subtilisin. The rPmSERPIN3 also inhibited the shrimp prophenoloxidase system activation in vitro. Injecting the rPmSERPIN3 along with Vibrio harveyi into the shrimp decreased the clearance rate of bacteria in the hemolymph. Potentially, the PmSERPIN3 functions as a regulator of the proPO activating system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) has previously been shown to have very active in vitro antimicrobial activity against a broad range of Gram-positive and Gram-negative bacteria, certain fungi and viruses, including known pathogens of P. monodon shrimp. With respect to the strong bactericidal effect on Gram-negative and Gram-positive bacteria, the ALFPm3 binds to their principal cell wall components, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), with a high affinity. The aim of this study was, therefore, to reveal the effects of treating ALFPm3 on membrane of Vibrio harveyi, a P. monodon pathogenic Gram-negative bacterium. The recombinant (r)ALFPm3 protein was found to localize on the V. harveyi cells in vivo, followed by inducing membrane permeabilization and leakage of cytoplasmic components. Moreover, the effect of rALFPm3 treatment on the bacterial cell morphology was confirmed by scanning and transmission electron microscopy. Membrane disruption and damage, bleb and pore formation, and the leakage of cytoplasmic contents were all clearly observed. Taken together, these results suggested that ALFPm3 effectively kills bacteria through bacterial membrane permeabilization., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides previously identified in various crustaceans. Out of five isoforms identified in Penaeus monodon, ALFPm3 is the best characterized, exhibits antibacterial and antifungal activities and can protect the shrimp from viral infections. Herein, the most recent identified ALFPm, called ALFPm6, is characterized for its potential role in the shrimp's immunity. RNA interference-mediated gene silencing was used to study the function of ALFPm6 in comparison to ALFPm3. Knockdown of ALFPm3 gene led to rapid death with a cumulative shrimp mortality of 86% within 7 days, accompanied by a 12- and 50-fold higher bacterial count after 2 days in the haemolymph and hepatopancreas, respectively, compared to the control shrimp injected with GFP dsRNA. In contrast, gene silencing of ALFPm6 alone had no effect on the shrimp mortality, but led to a significant increase in the cumulative mortality and a faster mortality rate following Vibrio harveyi and white spot syndrome virus (WSSV) infections, respectively. These results support the roles of ALFPm6 and ALFPm3 in the protection of shrimp against microbial infections., (Copyright © 2011 Elsevier Ltd. All rights reserved.)